Highly Aggressive Invasive Race Group PstS2 in Russian Populations of the Wheat Yellow Rust Pathogen

The expansion of the area of harmfulness of the wheat yellow rust pathogen (Puccinia striiformis) (Pst) has be observed all over the world in the 2000s. This is due to the emergence of new highly aggressive invasive groups of races PstS1 and PstS2, adopted to the high temperatures, and also as a result of virulence mutations of regional pathogen populations. SCAR-markers were developed for identification of invasive races, and pathogen collections from many countries were studied. In these studies in first in Russia, the analysis of regional populations of P. striiformis for the presence of invasive races PstS1 and PstS2 was carried out. Single pustule isolates were obtained from urediosamples collected from common and durum wheat, triticale and wild grasses in seven regions of the Russian Federation (North Caucasian, Northwestern, Central Black Earth, Lower Volga, Middle Volga, Volga–Vyatka, West Siberian) in 2019–2020. In total 82 isolates were studied. Using SCAR markers, three genotypes were identified in the studied collection of P. striiformis, and one of which belongs to the invasive group PstS2. The other two genotypes had a different origin (other). Isolates of PstS2 group were received from pathogen population samples collected in the Russian Northwest in 2020. Virulence analysis revealed two phenotypes among them: PstS2_R1 (3 isolates) and PstS2_R2 (1 isolate). According to information from the Global Rust Reference Center (http://www.wheatrust.org/), the main characteristic of isolates from invasive PstS2 group is virulence to wheat lines with resistance genes Yr2, Yr6, Yr7, Yr8, Yr9, and Yr25. The Russian R1 phenotype PstS2 was also characterized by virulence to these genes, as well as to Yr1, Yr32, and YrSp. The PstS2_R2 phenotype differed from PstS2_R1 for avirulence to Yr25 and virulence to Yr3 and Yr4. The main difference of Russian PstS2 isolates with detected in other countries is virulence to wheat lines with genes Yr4, Yr32, and YrSp. The first detection of invasive races in the Northwest of Russia indicates the relevance of annual monitoring of regional populations of P. striiformis.


INTRODUCTION
Wheat is the main strategic crop.Diseases are one of the factors reducing the yield of grain and the quality of its products [1].Rust is the most widespread disease of this crop.There are three species of rust pathogens occurring on wheat: Puccinia triticina Erikss., P. graminis Pers., and P. striiformis West.[2], which are characterized by high evolutionary potential.The accelerated rate of mutations predetermines the emergence of new races.Greater migratory propensity of the pathogens favors their rapid spread throughout countries and continents [3,4].
Brown rust had been economically significant for wheat until 2005 in most countries, including Russia [5,6].In the modern period, situation has changed and brown rust has been replaced by yellow and stem rusts.The yellow rust pathogen affects both cultivated and wild type cereals, including soft and hard wheat, triticale, barley and rye.The symptoms of the disease are observed on leaves, leaf sheaths, spikelet scales and, less frequently, on stems, as lemon yellow uredinio-pustules arranged in longitudinal rows [7].Therefore, another popular name of this disease is stripe rust.
Air humidity and temperature are the major factors that determine successful development of rust fungi.P. striiformis develops at lower temperatures (2-15°С) and higher atmospheric humidity [4], which limited wide distribution of the pathogen, in contrast to more plastic species P. triticina and P. graminis.
Uredinio-spores of the yellow rust pathogen in the winter period die at -4°С, while fungal mycelium in living tissue of a wheat leaf remains viable even at lower temperatures [8].J.W. Hendrix and E.H. Lloyd (1966) report that one uredinio-pustule per 1 ha of wheat is enough for epiphytotic development of the disease under favorable weather conditions in spring [9].In case of favorable weather conditions and early a All-Russian Institute of Plant Protection, St. Petersburg, 196608 Russia *e-mail: eshaydayuk@bk.ru**e-mail: eigultyaeva@gmail.comdevelopment of the disease, yield losses can be up to 100% [10].
Until recently, wheat yellow rust has been one of the diseases of regional significance worldwide.In the 2000s, its area and harmfulness began to increase substantially.Epiphytotics of the disease are regularly observed in West Europe, Central and East Asia, in the Middle East, North and South Africa, North and South America, Australia, Central Asia and Kazakhstan [4,[11][12][13][14][15][16][17].This is due to the appearance of new highly aggressive groups of races PstS1 and PstS2 that are able to develop at high temperatures.In addition, rapid mutations of the pathogen in virulence have been noted.The new races appearing in West Europe, Australia, and North America have overcome the resilience of previously resistant wheat and triticale varieties and now rapidly spread around the world [18,19].
The PstS1 race group was found for the first time in Kenia in 1982.Later, its presence was recorded in other countries of East Africa: Ethiopia (1986), Rwanda, Burundi (1988), Tanzania (1990).It was recorded for the first time in the United States in 2000 and in Australia in 2002, being the cause of the strongest epiphytotics of yellow rust in these countries [20][21][22][23].The difference between PstS1 and other globally widespread races is the adaptation to high temperatures, which is untypical of this pathogen [21,24,25].At present, the PstS1 group is annually detected in East Africa and, in the years of epiphytotics, in the United States and Australia (https://agro.au.dk/ forskning/internationale-platforme/wheatrust); how-  [23,[26][27][28].In 2015-2016, PstS2 isolates were detected in the countries bordering Russia: Ukraine and Azerbaijan.It is hypothesized that the emergence and distribution of new races PstS1 and PstS2 can be due to overcoming the efficiency of resistance gene Yr9 in the Middle East and in South Asia in the 1980s and 1990s [29].
The Russian populations of the yellow rust pathogen have not been studied for the presence of invasive races PstS1 and PstS2 to date.At the same time, both groups are characterized by the high rate of variability with respect to virulence and microsatellite loci, which determines the need for their monitoring.The present work is aimed at the molecular-genetic analysis of the Russian populations of P. striiformis for the presence of highly aggressive invasive groups of races PstS1 and PstS2.

MATERIALS AND METHODS
The infection samples represented by the leaves with P. striiformis uredinio-pustules were obtained from seven regions of the Russian Federation: North Caucasian (Dagestan, Krasnodar Krai), Northwestern (Leningrad Oblast), Central Black Earth (Tambov Oblast), Lower Volga (Saratov Oblast), Middle Volga (Samara Oblast), Volga-Vyatka (Kirov Oblast), and West Siberian (Novosibirsk Oblast, Krasnoyarsk Krai) in 2019-2020.They were collected from soft and hard wheat, triticale and cereal grasses on experimental fields, state variety plots (SVP) and industrial crops.The origin of infectious material is given in Table 1.
The results were interpreted in accordance with the following principle: the isolates belonging to the PstS1 group have amplification products of all four markers: SCP19M24 a1, 485 b.p.; SCP19M24 a2, 385 b.p.; SCP19M26a1, 491 b.p.; SCP19M26a2, 262 b.p; the isolates of the PstS2 group have amplification products of the SCP19M24a1, SCP19M24a2, and SCP19M26a2 markers.
Molecular analysis has shown three genotypes in the studied collection of P. striiformis (Table 2).According to Walter et al. (2016), isolates having diagnostic fragments with the SCP19M24a1, SCP19M24a2, and SCP19M26a2 markers belong to the PstS2 invasive group.Such P. striiformis isolates were detected in the samples of the Northwestern population collected in 2020 on the experimental field of Pushkin VIR Laboratories (St.Petersburg, Pushkin) (2 isolates) and Leningrad SVP (Gatchina District, Rozhdestveno and Volosovo).Two other genotypes differed from PstS1 and PstS2, which was indicative of their different origin.In global literature, such isolates are referred to as "other" [23].The examples of electrophoregrams are shown in Fig. 1.
The analysis of a vast collection of isolates at the Global Rust Reference Center has shown that isolates from PstS1 and PstS2 invasive groups have no specific differences in virulence and can be similar both to each other and to isolates not belonging to these groups [33].Worldwide, three and nine phenotypes of virulence have been determined in the PstS1 and PstS2 groups, respectively (Table 3).The most typical feature of isolates from both invasive groups is virulence to the samples with the genes Yr2, Yr6, Yr7, Yr8, Yr9, and Yr25 [21,28].The Russian phenotype PstS2_R1 represented by three isolates was characterized by virulence to all of these genes and, in addition, was virulent for Yr1, Yr32, and YrSp.The PstS2_R2 phenotype differed from PstS2_R1 in avirulence to Yr25 and virulence to Yr3 and Yr4.The main difference between the Russian isolates of PstS2 group and those identified in other countries is virulence to the lines with the genes Yr4, Yr32, and YrSp.

CONCLUSIONS
The molecular genetic analysis of populations of the wheat yellow rust pathogen for the presence of highly aggressive groups of races PstS1 and PstS2 was performed in Russia for the first time.Isolates belonging to the PstS2 group have been found in the samples of the Northwestern population collected in the Leningrad Oblast.Isolates of the invasive group demonstrate two phenotypes of virulence different from those identified in other countries.The first identification of invasive races in the territory of Northwestern Russia indicates the need for the annual monitoring of regional populations of P. striiformis.

Table 1 .
Characteristics of the studied collections of Puccinia striiformis

Table 2 .
CharacteristicsThe PstS2 group is a result of mutation in PstS1, i.e., in essence, is its sister line.In contrast to PstS1, it has become widespread.It was observed in the Middle East and in North Africa in the 1990s; in West Europe in 2002; in West and Central Asia in 2003 of the Puccinia striiformis genotypes determined using SCAR markers and their representation in the pathogen collection under study *Presence (+), absence (-) of amplification product.

Table 3 .
Virulence of the Puccinia striiformis isolates belonging to the PstS1 and PstS2 groups abroad (http://www.wheatrust.org/) and in Russia